Hepatitis A vaccines

ABSTRACT

A process for the production of inactivated Hepatitis A virus substantially free of host cell contamination is described, the process comprising: a)culturing Hepatitis A virus and harvesting a Hepatitis A preparation; b) treating said Hepatitis A preparation with a protease; and thereafter c) separating intact virus from protease-digested material; d) inactivating said virus. Also described are vaccines comprising the inactivated Hepatitis A virus, preferably in combination with strong adjuvants.

The present invention relates to new vaccine compositions, processes fortheir manufacture and their use in medicine. In particular, the presentinvention relates to improved Hepatitis A vaccines adjuvanted with apotent immunostimulator, preferably such as monophosphoryl lipid A or aderivative thereof The invention also relates to combination vaccines inwhich the Hepatitis A vaccine is a component.

Hepatitis A vaccines are known. For example the vaccine Havrix (TradeMark), from SmithKline Beecham Biologicals can be used to preventhepatitis A infections and is also formulated with aluminium hydroxideas adjuvant. This vaccine is produced according to the procedure ofAndre et al. It comprises an attenuated strain of the HM-175 Hepatitis Avirus inactivated with formol (formaldehyde); see Andre et al [Prog Med.Virol. 1990, vol 37; -p72-95].

The vaccine Twinrix (Trade Mark) which is a combination of the abovehepatitis A and hepatitis B antigens may be used to protect againstHepatitis A and Hepatitis B simultaneously. The vaccine Hepatyrix (TradeMark) which is a combination of the above hepatitis A antigen and aSalmonella typhimurium purified Vi polysaccharide may be used to protectagainst Hepatitis and typhoid simultaneously.

International patent application WO93/19780 (SmithKline BeechamBiologicals s.a.) discloses, inter alia, a Hepatitis A vaccineadjuvanted with 3D-MPL.

European patent 0 339 667 (Chemo Sero) describes the general concept ofcombining a hepatitis A antigen and a hepatitis B antigen to make acombination vaccine. In that specification it is stated that theadjuvant which is used is not critical: it must only be capable ofenhancing the immune activity to a desired extent and not cause any sideeffects. It is stated that aluminium gel may be used, in particularaluminium hydroxide gel and aluminium phosphate gel.

It has now been found that traditional processes for producing andpurifying inactivated virus for hepatitis A vaccines can leave a smallresidue of contaminants from the host cells in which the hepatitis Avirus was grown. Such host cell contaminants, especially when they arefrom human origin, diploid in nature and at a low level, provide noconcern when the vaccine is adjuvanted with aluminium salts. But whenthe vaccine is adjuvanted with strong immunostimulants there is atheoretical possibility that a vaccinee may raise an adverse immuneresponse to the host contaminants.

Accordingly there is a need for a method of manufacture which removessubstantially all traces of such host cell proteins.

Accordingly in one aspect of the invention there is provided a processfor the production of inactivated Hepatitis A virus substantially freeof host cell contamination, the process comprising:

-   -   a) culturing Hepatitis A virus and harvesting a hepatitis A        preparation;    -   b) treating said hepatitis A preparation with a protease; and        thereafter    -   c) separating intact virus from protease-digested material;    -   d) inactivating said virus.

Surprisingly, the protease digestion treatment does not adversely affectthe Hepatitis A virus, but facilitates the breakdown and separation ofhost cell contaminants from the Hepatits A preparation.

Preferably the Hepatitis A virus is derived from HM-175 strain.

By substantially free of host cell contamination is meant that less than10%, preferably less than 8%, more preferably less than 5% host cellprotein can be detected by scanning of silver-stained SDS PAGE. Moreimportantly and as determined by slot blot hybridisation one dose of HAVin the vaccine preferably contains less than 10 ng of host cellproteins.

Preferably the protease used is trypsin. Other proteases that may beutilised include pronase, papain, and pepsin.

The protease treatment is preferably carried out at above roomtemperature, e.g. at about 37° C. for about 2 hrs.

The separation of the intact virus from the protease and the digestedcomponents can be achieved by a variety of suitable methods, for exampleby permeation chromatography.

Alternatively the protease and digested components may be separated byany separation method that separates on the basis of size, for exampleultra filtration. The product can then be further purified by othersteps to remove other contaminants. For example, the product can befurther purified by subjecting the product to ion-exchangechromatography to remove any nucleic acid residue.

It is believed that the protease digestion step of the method accordingto the invention can improve purification of the hepatitis A preparationdue to two effects. First, the protease digests any contaminating hostproteins such that they are easier to separate in the chromatographicseparation step that follows the protease treatment. Second, thedigestion of contaminating host proteins allows better separation ofother contaminating materials which would otherwise be associated withundigested host proteins, in particular nucleic acid, in the ionexchange step. It will be appreciated that these observed effects do notnecessarily limit the invention in any way.

In another aspect of the present invention there is provided aninactivated Hepatitis A virus substantially free of contaminating hostproteins, as defined above.

The inactivated hepatitis A virus may then be formulated into a vaccine.

Thus the invention provides in a further aspect a Hepatitis A vaccinecomprising an inactivated hepatitis A virus substantially free of hostcell contaminants.

Such a vaccine may advantageously include a suitable adjuvant. Suitableadjuvants include an aluminium salt such as aluminium hydroxide gel oraluminium phosphate, but may also be a salt of calcium, iron or zinc, ormay be an insoluble suspension of acylated tyrosine, or acylated sugars,cationically or anionically derivatised polysaccharides, orpolyphosphazenes.

Advantageously, the highly purified hepatitis A virus may be formulatedwith strong adjuvant systems. Thus in the formulation of the invention,it is preferred that the adjuvant composition induces an immune responsecomprising Th1 aspects.

In general terms, a Th1-type response is characterised by the productionof IFN-γ as opposed to a Th2-type response which is characterised by theproduction of cytokines such as IL-4, IL-5 and IL-10. The isotypicprofile of the humoral response can also be used as a marker for Th1 orTh2-type responses. In mice Th1-type responses are often associated withthe generation of antibodies of the IgG2a subtype while IgG1 are markersof a Th2-type response. The situation is not as clear in humans but datasuggest that IgG1 and IgG4 could respectively be markers of Th1- andTh2-type responses.

Suitable adjuvant systems include for example a combination ofmonophosphoryl lipid A, preferably 3-O-de-acylated monophosphoryl lipidA (3D-MPL), and preferably formulated together with an aluminium salt.

An enhanced system involves the combination of monophosphoryl lipid Aand a saponin derivative particularly the combination of QS21 and 3D-MPLas disclosed in WO 94/00153, or a less reactogenic composition where theQS21 is quenched with cholesterol as disclosed in WO 96/33739.

A particularly potent adjuvant formulation involving QS21, 3D-MPL andd,1-alpha-tocopherol in an oil in water emulsion is described in WO95/17210.

Other known adjuvants which may be included are CpG containingoligonucleotides for example as disclosed in WO 96/02555.

Accordingly in a preferred embodiment of the present invention there isprovided a vaccine comprising a virus of the present invention,adjuvanted with monophosphoryl lipid A or a derivative thereof.

Preferably the vaccine additionally comprises a saponin, more preferablyQS21.

Preferably the formulation additionally comprises an oil in wateremulsion and d,1-alpha-tocopherol.

The present invention also provides a method for producing a vaccineformulation comprising mixing a purified virus of the present inventiontogether with a pharmaceutically acceptable excipient or carrier, suchas 3D-MPL.

The purified virus of the invention may advantageously be combined withother antigens so that it is effective in the prophylaxis or treatmentof other diseases in addition to hepatitis A infections. A preferredcombination involves a combination containing a hepatitis B antigen.

The preparation of Hepatitis B surface antigen (HBsAg) is welldocumented. See, for example, Harford et al in Develop. Biol. Standard54, page 125 (1983), Gregg et al in Biotechnology, 5, page 479 (1987),EP-A-0 226 846, EP-A-0 299 108 and references therein.

As used herein the expression ‘Hepatitis B surface antigen’0 or ‘HBsAg’includes any HBsAg antigen or fragment thereof displaying theantigenicity of HBV surface antigen. It will be understood that inaddition to the 226 amino acid sequence of the HBsAg S antigen (seeTiollais et al, Nature, 317, 489 (1985) and references therein) HBsAg asherein described may, if desired, contain all or part of a pre-Ssequence as described in the above references and in EP-A-0 278 940. Inparticular the HBsAg may comprise a polypeptide comprising an amino acidsequence comprising residues 12-52 followed by residues 133-145 followedby residues 175-400 of the L-protein of HBsAg relative to the openreading frame on a Hepatitis B virus of ad serotype (this polypeptide ifreferred to as L*; see EP 0 414 374). HBsAg within the scope of theinvention may also include the preS1-preS2-S polypeptide described in EP0 198 474 (Endotronics) or analogues thereof such as those described inEP 0 304 578 (McCormick and Jones). HBsAg as herein described can alsorefer to mutants, for example the ‘escape mutant’ described in WO91/14703 or EP 0 511 855 A1, especially HBsAg wherein there is an aminoacid substitution at position 145 to arginine from glycine.

Normally the HBsAg will be in particle form. The particles may comprisefor example S protein alone or may be composite particles, for example(L*,S) where L* is as defined above and S denotes the S-protein ofHBsAg. The said particle is advantageously in the form in which it isexpressed in yeast.

The invention in a further aspect provides a vaccine formulation asdescribed herein for use in medical therapy, particularly for use in thetreatment or prophylaxis of hepatitis viral infections. In a preferredaspect the vaccine accordingly to the invention is a therapeutic vaccineuseful for the treatment of ongoing hepatitis infections, moreespecially hepatitis A and/or hepatitis B infections in humans sufferingtherefrom.

In view of the surprisingly efficacious results obtained, in a furtherpreferred aspect the invention provides a vaccine composition for thetreatment or prophylaxis of Hepatitis A and/or Hepatitis B infections.

Advantageously the hepatitis vaccine composition of the inventioncontains other antigens so that it is effective in the treatment orprophylaxis of one or more other bacterial, viral or fungal infections.

Accordingly the hepatitis vaccine formulation according to the inventionpreferably contains at least one other component which may be selectedfrom non-hepatitis antigens which are known in the art to affordprotection against one or more of the following diseases:

-   -   diphtheria, tetanus, pertussis, Haemophilus influenzae b (Hib),        and polio.

Preferably the vaccine according to the invention includes HBsAg ashereinabove defined.

Suitable components for use in such combination vaccines are alreadycommercially available and details may be obtained from the World HealthOrganization. For example the polio component may be the Salkinactivated polio vaccine (IPV). The pertussis vaccine component maycomprise a whole cell or acellular product.

Advantageously the hepatitis or combination vaccine according to theinvention is a paediatric or an adolescent vaccine.

Preferred combination vaccines according to the invention for adolescentuse include one or more components selected from antigens which areknown in the art to provide protection against one or more of thefollowing diseases:

-   -   human papillomavirus (HPV), herpes simplex virus (HSV), Epstein        Barr virus (EBV), Varicella Zoster virus (VZV), human        cytomegalovirus (HCMV), Toxoplasma gondii.

The amount of each antigen in the vaccine dose is selected as an amountthat induces an immunoprotective response without significant adverseside effects in typical vaccinees. Such amount will vary depending onwhich specific immunogens are employed. Generally it is expected thateach dose will comprise 0.01 to 1.0 μg protein/dose for Hepatitis A,most preferably from between 0.06 to 0.220 μg protein/dose. For antigensother than Hepatitis A, for example HBsAg, HSV etc, the amount ofprotein per dose may be higher, e.g. up to about 20 μg per dose. Anoptimal amount of each of one or more immunogens for a particularvaccine can be ascertained by standard studies involving observation ofantibody titres and/or other responses in subjects. Following an initialvaccination, subjects may receive a boost within about 4 weeks.

In a further aspect of the present invention there is provided a methodof manufacture of a vaccine effective in preventing or treatinghepatitis infection, wherein the method comprises mixing the hepatitisantigen as defined herein with MPL or a derivative thereof.

Using this method one or more additional components are preferablyadmixed with the inactivated Hepatitis A vaccine to provide acombination vaccine.

The following examples illustrate the invention and its advantages.

EXAMPLES Example 1

a) Purification Steps

The series of steps given in this Example may be performed in differentcombinations in accordance with the method according to the invention,but always involving a trypsin or other protease digestion step.

Culture and Harvest

Hepatitis A virus HM175 is cultured on MRC5 cells (Andre et al supra)and the virus is harvested after washing of the cell layer to removeserum used in growth media. After freeze/thaw a detergent (Tween 20) isadded to extract the virus from the cell debris. Cell debris is removedby filtration through a 0.22 μm membrane. Filtrate is further subjectedto ultra-filtration. The resulting concentrate can eventually beclarified by centrifugation at 5-10,000×g for 1-2 hours.

Trypsination

The concentrate containing the HAV virus is treated with purifiedtrypsin extracted from pig pancreas. The trypsin used is doublecrystallised and kept frozen before use. Before addition of trypsin theconcentrate is prewarmed at 37° C. under constant agitation. Trypsin isthen added at a ratio of 440 IU per ml of concentrate, and the mixturegently stirred for minimum 2hrs at 37° C. (maximum 2.5hrs).

Concentration

After trypsin treatment the product can be processed without delay atambient temperature on an ultrafiltration device in order to reduce thevolume. The membrane used is regenerated cellulose with nominal cut-offof 30,000 Dalton, and up to a maximum of 8 ml of trypsinated product percm² of membrane, is processed at a transmembrane pressure between 0.2and 0.6 bar to achieve a concentration factor of between 8 and 12.

Permeation Chromatography

The aim of this step is to separate proteins from the intact HAV virus.When a permeation chromatography step is conducted after trypsintreatment, conditions have to be adapted to eliminate residual trypsinas well. The separation gel used is Permeation Sepharose 4BFF.

The virus is eluted at a smaller retention volume than the smallerprotein fragments which are eluted with larger retention volume (closerto the total volume of the column).

Chromatography parameters are as follows:

-   -   Chromatographic medium: Sepharose 4B FF (from Pharmacia)    -   Injected volume: 1 to 5% of gel volume    -   Elution rate: 5-10 cm/h    -   Temperature: 10 to 16° C.    -   Pool of fractions: target 100 ng prot/720 Elisa units (±10%)        Ion Exchange

The purpose of this purification step is to reduce the DNA content(originating from MRC5 cells). This step is run according to the batchprinciple.

The pool from the previous chromatographic step is adjusted to 0.3M NaCland then mixed with the ion exchange resin under mild agitation for 1 hr(maximum 1.5 hr) at room temperature.

After DNA fixation the gel is eliminated by filtration. The unfixed HAVvirus suspension is then diluted to adjust the NaCl concentration to 150mM. Alternatively, the ion exchange purification step can also beconducted by column chromatography.

The final purified product is sterile filtered on 0.22 μm filter.Chromatographic parameters are as follows:

-   -   Load: 3% of gel compared to the volume of the pool (vol/vol).    -   Temperature: ambient

Inactivation is carried out as described in Andre et al, except that 250μg/ml of formol is used.

Formaldehyde Reduction

Within 48 hr after the end of the inactivation the product isdiafiltered and concentrated in order to reduce the formaldehyde contentand to be preadsorbed on an aluminium salt (preferably aluminiumhydroxide or aluminium phosphate).

Prior to use, the complete ultrafiltration device is sanitised with 0.1NNaOH for at least 30 minutes. The device is then thoroughly rinsed withdiafiltration buffer and the membranes are then coated with a buffercontaining amino acids (Travasol). Finally the device is rinsed withdiafiltration buffer.

After diafiltration and concentration the final product is sterilefiltered on a 0.22 μm filter.

b) Purification Schemes

The purification steps described above were combined in such a way thata pure product was obtained in an economical way. Two such purificationschemes are presented in Scheme 1, both of which yield a similarproduct. In one configuration of the steps as shown in scheme 1, thesteps were carried out in the order described in Example 1a, and in theother configuration the trypinisation step was carried out between theultrafiltration and first permeation chromatography steps. This meantthat the second permeation chromatography step could be eliminated.

Example 2 Characterisation

Samples of purified product were analysed by SDS PAGE 12.5% acrylamide,1% SDS in a stacking gel, migration for 15 h at 45-50 volts. The gel wasstained with AgNO₃ and the colour was allowed to develop for 10 to 20min and compared with traditional HAV processes (Andre et al).

As can be seen from FIGS. 2 and 3, by subjecting the product to proteasetreatment a majority of high molecular weight contaminants are removed.

Example 3 HAV Vaccine Formulations

3.1 HAV−alum 3D-MPL

The HAV particle of example 1 was first adsorbed on to aluminiumhydroxide (superfos) followed by the addition of free 3D-MPL. A 0.5 mldose 720 ELU of Hepatitis A virus particle/0.025 mg A1³⁺ ion and 50 μgof 3D-MPL.

3.2 HAV+Hbs Ag Formulations

The following formulations were made:

-   -   1. Hep B S Ag 20 μg/AlP0₄+HA 720/Al(OH)₃    -   2. Hep B S Ag 20 μ/AlPO₄/3D-MPL 50 μg+HA 1440/Al(OH)₃    -   3. Hep B S Ag 20 μg/AlPO₄+3D-MPL 50 μg/Al(OH)₃+HA 720/Al(OH)₃    -   4. Hep B S Ag 20 μg/AlPO₄+3D-MPL 50μg/AlPO₄+HA 720 Al(OH)₃    -   5. Hep B S Ag 20/μg/AlPO₄/3D-MPL 50 μg+HA 720/Al(OH)₃

In group 1 the individual antigens were adsorbed on to the aluminiumsalt 0.025 mg Al³⁺ ion (Al(OH)₃ Superfos) for HA, 0.475 mg Al3+ion(AlPO₄ Superfos type). In group 2 and 5, 50 μg/dose of free 3D-MPL wasadded to adsorbed Hepatitis S antigen to which the adsorbed hepatitis Acomponent was added. In group 3 and 4, 3D-MPL was separately adsorbed onto the aluminium salt, and then the three adsorbed components were mixedtogether.

Example 4 Immunogenicity Experiments

Balb/c Mice

Groups of 10 mice were immunised intramuscularly three times at 2 weeksinterval with HAV/HBs formualtions (1/10 HD). Antibody response to Hbswere monitored by ELISA at 14 days post II and 14 days post III. Theisotypic profile of the anti-HBs response was analysed at 14 days postII. Antibody response to HAV was monitored 14 days post III.

NMRI Mice

Groups of 10 mice were immunised intraperitoneally once with HAV/HBsformulations (1/2 HD). Antibody response to Hbs and HAV were monitoredby ELISA at 28 days post injection. Formulations Group Vaccine lotFormulation 1 HAB112B6 HBs 20 μg/AlPO4 + HAV 720/Al(OH)3 2 DHAB713 HBs20 μg/AlPO4/MPL 50 + HAV 1440/Al(OH)3 3 DHAB717 HBs 20 μg/AlPO4 + MPL50/Al(OH)3 + HAV 720/Al(OH)3 4 DHAB718 HBs 20 μg/AlPO4 + MPL 50/AlPO4 +HAV 720/Al(OH)3 5 DHAB716 HBs 20 μg/AlPO4/MPL 50 + HAV 720/Al(OH)3HAV Mouse Serology

Quantitation of anti-Hepatitis A Virus antigen (HAV) antibody wasperformed using Enzygnost kit from Behring (ref: OQEC11). This assay isan ELISA based on the competitive test principle, run in one step andinitially developed for human serology.

Two-fold dilution of mice sera (4 dilutions starting at 1/10) humananti-HAV reference (8 dilutions starting at 80 mlU/ml) and controls wereperformed in anti-HAV negative human sera. Mixtures of test/controlsamples (25 μl), HAV antigen solution (50 μl) and anti-HAV mousemonoclonal conjugated with peroxidase (50 μl of 1/41 dilution performedin conjugate buffer) were incubated on HAV pre-coated microplates for 2hrs at 37° C. The plates were then washed and incubated for 30 min witha solution of TMB (100 μl). The reaction was stopped with H₂SO₄ 0.5N andread at 450/620 nm.

Anti-HAV antibody titers were calculated from the reference bySoftmaxPro (using a four parameters equation) and expressed in mlU/ml.

Results

The results are shown in FIG. 1.

In FIG. 1 a results demonstrate that formulations containing MPL inducesignificantly higher antibody responses to the hepatitis A componentthan the aluminum salt group alone. Similarly the results shown in FIG.1b demonstrate that MPL containing formulations induce higher antibodytitres to HbsAg.

Example 5 Clinical Studies

HAV/HBs (HAB) formulations were administered to healthy subjects.

Serum titres of anti-HAV antibodies were measured by ELISA (Enzymun testfrom Boehringer Mannheim) and anti-HBs antibodies by radioimmunoassay(RIA) using test kit AUSAB-Abbott. The assay cut-off for anti-HAVantibodies was 33 mIU/ml and the assay cut-off for anti-HBs antibodieswas 1 mIU/ml.

Subjects with anti-HAV antibody titres of 33 mIU/ml were considered tobe seropositive for anti-HAV antibodies. Subjects with anti-HBs antibodytitres 1 mIU/ml were considered to be seropositive for anti-HBsantibodies. Seroprotection rate for anti-HBs was defined as the ratio ofsubjects with anti-HBs titres 10 mIU/ml. Vaccine lots CommercialCombined HAB/MPL TwinrixTM (adult) candidate Group 1 Group 3 Lot n^(o)HAB 116C4/M1 DHAB 713A2 Inactivated hepA at least 720 EL.U at least 1440(new process) (strain HM175-RIT EL.U 4380) Recombinant HBsAg 20 μg 20 μgMPL — 50 μg Al salt 0.45 mg 0.5 mg Volume/dose 1.0 ml 0.5 mlResults:

In this phase I clinical trial where HAB/MPL was administered to healthysubjects, there is a marked effect of MPL acting as immunostimulant onthe immune response.

MPL has a clear effect on anti-HAV kinetics. It induces a faster andstronger immune response with a marked anamnestic response observed atMth 6.5 and Mth 7 (i.e. 14 and 30 days respectively, after the lastvaccine dose).

Within the limitations of the study, it can be concluded that thecandidate HAB/MPL vaccine exhibited a very good safety andreactogenicity profile. It was very immunogenic after a full vaccinationcourse of 2 doses in the study cohort of healthy adults aged 18-40years. There was a strong priming and faster immune response to both hepA & B antigens. 1. Anti-HAV Kinetics D7 D9 D11 D13 D15 Twinrix 720/20 SC%  5 10 30  65  74 0, 1, 6 M GMT 35 41 71  84 176 N = 20 1440 HAV (newprocess) SC % 10 60 95 100 100 20 μg HBsAg GMT 37 43 125  316 569 50 μgMPL (mixed) 0.5 mg Alum - 0, 6M N = 20 2. Anti-HAV Titers D15 M1 M7Twinrix 720/20 GMT 176 349  7107 0, 1, 6 M N = 50 1440 HAV (new process)GMT 569 888 13386 20 μg HBsAg 50 μg MPL (mixed) 0.5 mg Alum - 0, 6M N =50

Scheme 1 PURIFICATION PROCEDURES FOR HAV 175 strain HARVEST ⇓ THAWINGADDITION TWEEN 20 CLARIFICATION (0.22 μm) ⇓ ULTRAFILTRATIONULTRAFILTRATION (cut-off 300.000) (cut-off 300.000) ⇓ ⇓ TRYPSINISATIONCHROMATOGRAPHY (440 UI/ML - 2 h - 37° C. (permeation Sepharose 4BFF) ⇓ ⇓ION EXCHANGE POOL (DEAE Sepharose 6B) ⇓ ⇓ TRYPSINATION CHROMATOGRAPHY(440 UI/ML - 2 h - 37° C.) (gel permeation Sepharose 4BFF) ⇓ ⇓RECONCENTRATION FILTRATION ⇓ (0.22 μm) (CUT-OFF 30.000) ⇓ CHROMATOGRAPHY(permeation Sepharose 4BFF) ⇓ POOL ⇓ ION EXCHANGE (DEAE Sepharose 6B) ⇓FILTRATION (0.22 μm) ⇓ INACTIVATION ⇓ FORMALDEHYDE REDUCTION andCONCENTRATION (cut-off 30.000) ⇓ STERILE FILTRATION ⇓ PREADSORPTION ⇓STORAGE ⇓ FORMULATION ⇓ FILLING

1-16. (canceled)
 17. An inactivated Hepatitis A virus substantially freeof host cell contaminants.
 18. The inactivated Hepatitis A virus ofclaim 17 comprising less than 10% contaminating host cell proteinsdetectable by scanning SDS PAGE.
 19. A Hepatitis A vaccine comprising aninactivated Hepatitis A virus according to claim
 17. 20. A vaccineaccording to claim 19 formulated with a Th1-type inducing adjuvant. 21.A vaccine according to claim 20, wherein the adjuvant comprisesmonophosphoryl lipid A or a derivative thereof.
 22. A vaccine accordingto claim 21, further comprising QS21.
 23. A vaccine according to claim22, further comprising an oil in water emulsion and tocopherol.
 24. Avaccine according to claim 18, further comprising a Hepatitis B antigen.25. A vaccine according to claim 22, further comprising a Hepatitis Bantigen.
 26. A vaccine according to claim 23, further comprising aHepatitis B antigen.
 27. A vaccine according to claim 18, furthercomprising a non-hepatitis antigen.
 28. A vaccine according to claim 22,further comprising a non-hepatitis antigen.
 29. A vaccine according toclaim 23, further comprising a non-hepatitis antigen.
 30. A vaccineaccording to claim 24, further comprising a non-hepatitis antigen.
 31. Avaccine according to claim 25, further comprising a non-hepatitisantigen.
 32. A vaccine according to claim 26, further comprising anon-hepatitis antigen.
 33. The virus of claim 17 wherein the virus isobtained by a process comprising: a) culturing Hepatitis A virus andharvesting a hepatitis A preparation; b) treating said hepatitis Apreparation with a protease, thereafter; c) separating intact virus fromprotease-digested protein; and d) inactivating said virus.
 34. AHepatitis A vaccine comprising an inactivated Hepatitis A virusaccording to claim 33.